1. Field of the Invention
The invention relates generally to the field of heart disease and cardiovascular disease. More specifically, the invention is directed to a method of decreasing atheroma formation in mammals, by administration of paraoxonase-1 (PON-1), an expressed protein that has hydrolase activity for organophosphates, and antioxidation activity for low-density lipoprotein (LDL).
2. Background
It is by now well-accepted that atherogenesis and hyperlipidemia are intimately related. Atherogenesis involves the build-up of cholesterol within the endothelium of arterial walls and the subsequent formation of placques. Placques can fissure, ultimately causing thrombus formation which may lead to stroke or myocardial infarction. Of the two forms of lipoproteins, high-density (HDL) and low-density lipoprotein (LDL), LDL is positively correlated with placque formation, while HDL is thought to be anti-atherogenic through the reverse cholesterol transport mechanism (see below).
The lipid transport system is divided into two major pathways, the exogenous pathway (dietary triglycerides and cholesterol absorbed by the intestine) and the endogenous pathway (triglycerides and cholesterol secreted by the liver). The reverse cholesterol transport system, mediated by HDL, is involved in both pathways and is thought to be a major non-receptor based mechanism for removal of cholesterol by HDL. Two subsets of HDL are involved in reverse cholesterol transport, HDL2 and HDL3. Nascent HDL accumulates cholesterol from cell membranes. The circulating enzyme lecithin-cholesterol acyltransferase (xe2x80x9cLCATxe2x80x9d) associates with HDL and esterifies free cholesterol, causing the esterified cholesterol to move into the core. HDL3 particles accumulate cholesteryl ester, and as it accumulates HDL3 becomes HDL2, which is rich in cholesteryl ester. The cholesteryl ester in HDL2 is then exchanged for triglyceride with the aid of cholesteryl ester transfer protein, converting HDL2 back to HDL3, which is then able to accumulate more free cholesterol. HDL is thought to be antiatherogenic through the reverse cholesterol transport system, because of its ability to take up excess free cholesterol.
Oxidation of LDL is a key intermediate in the formation of atherogenic placques. It has been found that LDL must undergo modification before it can be ingested by macrophages to form foam cells, which are important components of atherosclerotic placques (Steinberg, D., et al., xe2x80x9cBeyond cholesterol: modifications of low-density cholesterol that increase its atherogenicity,xe2x80x9d N. Engl. J. Med. 320:915 (1989)). In vivo, oxidation is probably the most frequent form of LDL modification. Oxidized LDL not only contributes to the formation of foam cells, but also is chemotactic for circulating monocytes, is cytotoxic, and impairs endothelial function.
HDL was found to inhibit LDL oxidation, which is another potential mechanism by which HDL may reduce atherosclerosis. S. Parthasarathy and coworkers have shown that incubation of HDL with oxidatively-modified LDL results in inhibition of production of thiobarbituric acid-reactive products (TBARS) (Parthasarathy, S. et al., xe2x80x9cHigh-density lipoprotein inhibits the oxidative modification of low-density lipoprotein,xe2x80x9d Biochim. Biophys. Acta 1044:275 (1990)). However, the mechanism for HDL""s antioxidative function remains unknown. In another study, Klimov et al. injected 200 mg of human HDL3 into rabbits which had been rendered hypercholesterolemic by cholesterol feeding. Total plasma conjugated dienes and trienes were reduced by 20-30% six hours after the injection and remained at that reduced level up to twenty-four hours after the injection (Klimov, A. N., et al., xe2x80x9cAntioxidative activity of high density lipoproteins in vivo,xe2x80x9d Atherosclerosis 100:13 (1993)).
Antioxidant therapy has been shown to improve endothelial cell function in patients with hypercholesterolemia and coronary artery disease (Anderson, T J, et al., xe2x80x9cThe effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion,xe2x80x9d N. Engl. J. Med. 332:488 (1995)). The Cambridge Heart Antioxidant Study (CHAOS) randomized 2,002 patients with proven coronary disease to vitamin E, 400 to 800 I.U., or placebo. After a median follow-up of 1.4 years, antioxidant treatment reduced the primary endpoint of cardiovascular death and nonfatal MI by 47 percent (41 v. 64 events) (Stephens, N. G., et al., xe2x80x9cRandomized controlled trial of Vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS),xe2x80x9d Lancet 347:781 (1996)).
Paraoxonase (PON) is a protein secreted by the liver that is found primarily in serum. The name is derived from its ability to hydrolyze the organophosphate paraoxon in vivo. There are 3 known allelic forms of PON. Serum paraoxonase/arylesterase (PON-1) is a 354 residue 43-45 kDa A-esterase associated with HDL (Kelso, G. J., et al., xe2x80x9cApolipoprotein J is associated with paraoxonase in human plasma,xe2x80x9d Biochemistry 33: 832-839 (1994)). It is well-known to be involved in the hydrolysis of several organophosphate insecticides (Murphy, S. D. in Toxicology: The Basic Science of Poisons, (eds. Doull, J., Klassen, C., and Amdur, M.) 357-408, Macmillan, New York, (1980); Tafuri, J., et al., xe2x80x9cOrganophosphate poisoning,xe2x80x9d Ann. Emerg. Med. 16:193-202 (1987)). PON2 and PON3 are known allelic variants that have similar sequences. It is not known if PON2 or PON3 are expressed in vivo. U.S. Pat. Nos. 5,792,639 and 5,629,193 (Human Genome Sciences) are directed to a human paraoxonase gene, its associated vectors and transformed host cells and their use to detoxify organophosphates in vivo and for a neuroprotective effect. The DNA sequence claimed by HGS is likely that of PON2 based on homology searching. An alignment of the PON1 and PON2 nucleic acid sequences shows 69% identity. There is no suggestion in either the ""639 or the ""193 patents for the use of paraoxonase to reduce atheroma formation described herein.
The physiologic activity of the PON family members was, until recently, unknown. It has recently been postulated that PON may play a role as an in vivo antioxidant that may reduce the peroxidation of LDL (Mackness, M. I., et al., xe2x80x9cHDL, its enzymes and its potential to influence lipid peroxidation,xe2x80x9d Atherosclerosis 115:243-253 (1995)). However, the same review stated that other enzymes resident on HDL may also play the same role, such as platelet activating factor acetylhydrolase (Stafforini, D. M., et al., xe2x80x9cThe plasma PAF acetylhydrolase prevents oxidative modification of low density lipoprotein,xe2x80x9d J. Lipid Mediators Cell Signaling 10:53 (1994)).
Several human population studies have revealed significant associations between the common polymorphisms of the PON1 gene and coronary artery disease (CAD) (Ruiz, J., et al., xe2x80x9cGln-Arg192 polymorphism of paraoxonase and coronary heart disease in type 2 diabetes,xe2x80x9d Lancet 346: 869-872 (1995); Serrato, M., et al., xe2x80x9cA variant of human paraoxonase/arylesterase (HUMPONA) gene is a risk factor for coronary artery disease,xe2x80x9d J. Clin. Invest. 96: 3005-3008 (1995)). Also, PON-1 has the capacity to destroy certain proinflammatory oxidized phospholipids found in oxidized LDL (Mackness, M. I., et al., xe2x80x9cParaoxonase prevents accumulation of lipoperoxides in low-density lipoprotein,xe2x80x9d FEBS Lett 286: 152-154 (1991); Watson, A. D., et al., xe2x80x9cProtective effect of HDL associated paraoxonase-inhibition of the biological activity of minimally oxidized low density lipoprotein,xe2x80x9d J. Clin. Invest. 96: 2882-2891 (1995)). Again, there has been no isolation of the mechanism except to suggest that paraoxonase may be involved.
Mackness et al. (xe2x80x9cIs Paraoxonase related to Atherosclerosis,xe2x80x9d Chem.-Biol. Interactions 87:161-171 (1993)) discuss the evidence for an anti-oxidative role for paraoxonase. In this paper they investigated the serum paraoxonase activity in two populations prone to developing atherosclerosis, patients having familial hypercholesterolemia (FH) and IDDM (insulin-dependent diabetes mellitus). They showed a statistically significant increase in the percentage of the population in the low paraoxonase activity group in both FH and IDDM, two diseases manifesting a high occurrence of atherosclerosis. In addition, Mackness et al. studied, in an in vitro LDL oxidation model, the possible role of paraoxonase by adding small amounts in the presence of LDL under oxidative conditions. They concluded that paraoxonase is 300 times more active in preventing LDL oxidation than is HDL or its subfractions. However, they conclude that how paraoxonase protects LDL against oxidation in this model has yet to be determined, and several possibilities are discussed.
In genetic studies with mice, PON1 mRNA and protein levels correlate inversely with aortic lesion size (Shih, D. M., et al., xe2x80x9cGenetic-dietary regulation of serum paraoxonase expression and its role in atherogenesis in a mouse model,xe2x80x9d J. Clin. Invest. 97: 1630-1639 (1996)). These data suggest that PON-1 activity may bear some relationship to HDL levels and CAD observed in population studies (Tall, A., xe2x80x9cPlasma high density lipoproteins: Metabolism and relationship to atherogenesis,xe2x80x9d J. Clin. Invest. 86: 379-384 (1990)).
Familial hypercholesterolemia is a genetic disorder that results in chronically high levels of serum cholesterol, including both HDL and LDL. The disorder is also characterized as an LDL receptor defect. It is autosomal dominant with prevalence estimates of 1 homozygote per million of population. The LDL receptor normally participates in the uptake and subsequent elimination of LDL by hepatocytes. Accumulation of LDL in these patients is a result of the LDL receptor defect. There are definite high-incidence populations due to a founder effect, with French Canadians being the best known. The heterozygotes develop xanthomas at 20-30 years with atherosclerotic heart disease by 40-50 years in males and 50-60 years in females. Homozygotes usually do not survive beyond their thirties, due to cardiac infarctions caused by excessive placque accumulation. They have total cholesterol in the 500-1,000 mg/dl range, develop xanthomas by age 6, and develop symptomatic coronary artery disease by age 10.
Treatment of LDL receptor deficient patients is problematic. Homozygotes do not respond to HMG-CoA reductase inhibitors (they have no functional LDL receptors to upregulate), and heterozygotes respond half as well as normals. Niacin is effective in lowering LDL, but is poorly tolerated. Non-pharmacologic treatment includes weekly plasmapheresis, partial ileal bypass, protocaval shunts, and liver transplants.
There is a clear need for alternative treatments for those patients exhibiting hypercholesterolemia, particularly familial hypercholesterolemia.
This invention is directed to a method of decreasing atheroma formation in a mammal comprising a pharmaceutically effective amount of PON-1 or its functional equivalent. It is shown herein in an animal model that, surprisingly, PON-1 can act to reduce the area of aortic lesions, which are predictive of future atherosclerotic placques (atheromas). This discovery represents a potential pharmacological treatment for FH that should be beneficial for hypercholesterolemia generally.
It is an object of the invention to provide a method for decreasing atheroma formation in a mammal by administering a pharmaceutically effective amount of PON-1 or its functional equivalent, thereby decreasing the potential for atherosclerosis.
It is another object of the present invention to provide a new treatment for those afflicted with FH.